trichostatin-a and Carcinoma--Transitional-Cell

Topics: Animals; Carcinoma, Transitional Cell; Cell Line, Tumor; Cell Proliferation; Cell Survival; Drug Synergism; Gene Expression Regulation, Neoplastic; Histone Deacetylase Inhibitors; Humans; Hydroxamic Acids; MAP Kinase Signaling System; Mice; Paclitaxel; Treatment Outcome; Urinary Bladder Neoplasms; Xenograft Model Antitumor Assays

More than 14,000 people die from invasive urothelial carcinoma (iUC) of the urinary bladder each year in the USA, and more effective therapies are needed. Naturally occurring canine iUC very closely resembles the disease in humans and serves as a highly relevant translational model for novel therapy of human iUC. Work was undertaken to identify new targets for anticancer therapy in dogs with the goal of translating successful therapeutic strategies into humans with iUC.. Microarray expression analyses were conducted on mRNA extracted from canine normal bladder (n = 4) and iUC tissues (n = 4) using Genome Array 1.0 and analyzed by GeneSpring GX 11, with the stringency of P < 0.02 and a ≥ 2-fold change. The genes thus identified were further analyzed for functional and pathway analysis using Protein ANalysis THrough Evolutionary Relationships (PANTHER) Classification System. In selecting genes for further study, consideration was given for evidence of a role of the gene in human iUC. From these analyses, DNA methyltransferase 1 (DNMT1) was selected for further study. Immunohistochemistry (IHC) of canine normal bladder and iUC tissues was performed to confirm the microarray expression analyses. The effects of targeting DNMT1 in vitro was assessed through MTT assay and Western blot of canine iUC cells treated with 5-azacitidine (5-azaC) and trichostatin A (TSA).. DNMT1 was expressed in 0 of 6 normal canine bladder samples and in 10 of 22 (45%) canine iUC samples. The proliferation of canine iUC cells was inhibited by 5-azaC (at concentrations ≥ 5 μm) and by TSA (at concentrations ≥ 0.1 μm). Western blot results were supportive of DNMT1-related effects having a role in the antiproliferative activity.. Microarray expression analyses on canine tissues identified DNMT1 as a potentially "targetable" gene. Expression of DNMT1 in canine iUC was confirmed by IHC, and in vitro studies confirmed that drugs that inhibit DNMT1 have antiproliferative effects. These findings are similar to those recently reported in human iUC and are also in line with results of a preclinical (prehuman) trial of 5-azaC in dogs with naturally occurring iUC. DNMT1 has excellent potential as a target for iUC therapy in humans.

Topics: Animals; Antimetabolites, Antineoplastic; Azacitidine; Blotting, Western; Carcinoma, Transitional Cell; Cell Line, Tumor; Cell Proliferation; Cell Survival; DNA (Cytosine-5-)-Methyltransferase 1; DNA (Cytosine-5-)-Methyltransferases; Dogs; Dose-Response Relationship, Drug; Gene Expression Regulation, Neoplastic; Humans; Hydroxamic Acids; Immunohistochemistry; Oligonucleotide Array Sequence Analysis; Protein Synthesis Inhibitors; Reverse Transcriptase Polymerase Chain Reaction; Transcriptome; Urinary Bladder Neoplasms

Maspin is a member of serine protease inhibitor family with tumor suppressing activity for breast and prostate cancers, acting at the level of tumor invasion and metastasis. However, there have been no published data regarding the role of maspin in human bladder cancer. We evaluated maspin expression in 65 series of bladder cancer samples (22 transurethral resection (TUR) and 43 radical cystectomy) and studied the regulatory mechanism of maspin gene activation in bladder cancer cells. Maspin expression was immunohistochemically detected in four (18.2%) patients with TUR and 22 (51.2%) patients with radical cystectomy whereas no expression was observed in normal transitional cells located at tumor-free area in bladder. The maspin expression was significantly correlated with the development of muscle invasive bladder cancer (P=0.00008). Using a luciferase reporter system, maspin promoter activity was induced in the maspin-positive bladder cancer cell lines as well as maspin-negative RT4 cells. Furthermore, treatment with the DNA methyltransferase inhibitor, 5-aza-2' deoxycytidine, and histone deacetylase inhibitor, trichostatin A, led to re-expression of maspin in RT4 cells. Our results indicate that maspin may contribute to bladder cancer development and that DNA methylation and histone deacetylation may be important for regulating maspin gene activation in bladder cancer cells.

Topics: Adult; Aged; Azacitidine; Carcinoma, Transitional Cell; Cell Line, Tumor; Cytoplasm; Decitabine; DNA Methylation; Female; Gene Expression Regulation, Neoplastic; Genes, Reporter; Genes, Tumor Suppressor; Histones; Humans; Hydroxamic Acids; Immunohistochemistry; Luciferases; Male; Middle Aged; Promoter Regions, Genetic; Protein Biosynthesis; Proteins; Reverse Transcriptase Polymerase Chain Reaction; Serpins; Transcriptional Activation; Urinary Bladder Neoplasms